Process for preparing 5&#39;-nucleotides



United States Patent Ofiice 3,296,087 Patented Jan. 3, 196-7 The present invention relates to a process for preparing 5'-nucleotides, more particularly to a biochemical synthesis of 5-nucleotides.

It is an object of this invention to produce 5'-nucleotides economically on an industrial scale.

Such 5'-ribonucleotides as 5-inosinic acid, 5'-guanylic acid or 5'-Xanthylic acid are known to be useful as flavoring agents and also to possess useful pharmacological properties. Other nucleotides, such as 5'-adenylic acid, 5'- uridylic acid, and 5'-deoxyadenylic acid are of interest to pharmacologists and biochemists.

We have found that organic bases and 5'-ribonucleotides or 5'-deoxyn'bonucleotides derived from other bases react in the presence of certain bacteria to form the S- ribonucleotides or 5'-deoxyribonucleotides of the first mentioned organic bases in high yields.

The microorganisms which can be used in the present invention belong to many genera of bacteria. Suitable strains have been isolated from bacteria of the genera Pseudomonas, Alcaligenes, Serratia, Flavobacterium, Micrococcus, Staphylococcus, Proteus, Aerobacter and Escherichia. The following test has been employed for screening the bacteria.

Strains to be tested were cultured in a suitable conventional nutrient medium, and a suspension of living bacterial cells was prepared. The suspension containing 0.1-0.5 g. of cells, on a dry basis, was added to 100 ml. of a solution containing 0.135 'g. adenine and 1.43 g. disodium-5'-uridylate. The mixture was incubated without agitation at 40 C. for 3-20 hours While being kept mildly alkaline (pH 89), and the amount of 5'-adenylic acid formed was determined in a conventional manner by paper electro-phoresis in 10% acetic acid solution. Some 5'-inosinic acid may be formed by deamination of an amino group at the 6-position of 5-adenylic acid.

The following Table 1 shows the results of screening tests performed with various strains of bacteria.

TABLE 1 Amount of Amount of Yield of 5-adenylie 5-inosinic 5-nue1eo- Bacteria acid acid tide synthesized synthesized (percent) Achromobacter liquidum (IFO- 3084 O Achromobtlcter superficialie (1AM- 1433 0 0 Alcalignes metalcaligenea (AN-3) 0 1. 8 52.0 Alcaligenes viscolaclis (ATCC- 36) 0 1. 9 53. 7 Agrobacterium tumefaciens (ATCC-4720) 0 0 Agrobacterium radiobacter (ATC (3-6466) 0 0 Arthrobacter simpler (ATCC- 6496) 0 0 Arthrobacter ureafaciem (ATCC- 7562 0 0 Escherichia intermedia (A-21) 0. 2 0. l 5. 9 Escherichia coli HAM-1239) (ATOC-15289) 0- 1 0 2. 9 Aeromo'mzs hydrophz'la (1AM- 0. 1 0 Aerobacter cloacae (S-) 0 0 Erwi'm'a aroideae (IAM1068) 0 0 Erwihia carotovora (LAM-1024).--- 0 0 TABLE 1-Continued Yield of 5-nucleotide (percent) Amount of 5-inosinic acid synthesized a/ Amount of 5-adeny1ic acid synthesized a/ B acteria Xglghfmonas campestris (ATCC- 2 Cggyggbacterium faciens (1AM- corynebh'c'ziiiiiriifii'iiiiiii fiiasof Sarvina lutea (LAM-1099) Sarcimz albida (IAM-1012) Piiugomcmas trifolii (ATOC- 7) Pseudomonas trifolii (ATCC- 0. 1 0 Serrutia marcescens (ATCC- 14227) 0 Sermlia marcescens (ATCC- 2 Cilglomonas gelida (ATCC- BacizzaRHdil'i'sYiAiI-iibi')I:I: Bacillus megutherium (3-205-2) Vibn'o melsclmikovi (LAM-1039) Vibrio tyrogenes (IAM-lOSO) Flavobacterium harrisonii (A'ICC- Psiudomoms perlurida (ATCC- 1 53 Stzgalgflococcus citreus -(ATOC l Staphylococcus aureus (1AM- o o o 0 Q0000 Micrococcus varians (ATCC399) Migggoccus lysodeikticus (ATCC- Myco liifi'lihiiiiziiiK'i dd iisiI Mycoplona dimorpha (ATCC- CO 00 Doc loocoppppoooppoocooo I IFO: Identification number of the Institute for Fermentation, Osaka, I ZPJ Identification number of the Institute of Applied Microbiology, Tokyo University, Japan.

N, A, S, OM, YO: Identification numbers of the Institute for Food Microbiology, Chiba University, Japan.

ATCC: Identification number of the American Type Culture 001- lection, Washington, D.O.

Organic bases which have been used successfully in the method of the invention include purine bases, such as purine, adenine, hypoxanthine, Xanthine, guanine, 2- arninopurine, 2,6-diamino-purine, 6-dirnethy-laminopurine, benzyladenine, 6-methylamino-purine, or kinetin; and pyrimidine bases, such as uracil, S-hydroxymethyluracil, cytosine, thymine, or barbituric acid.

Suitable donors of ribose-5'-phosphate or deoxyribose- 5-phosphate radicals are 5-adenylic acid, 5-inosinic acid, 5-xanthylic acid, 5'-guanylic acid, 5-cytidylic acid, 5-uridylic acid, and other 5'-ribonucleotides; 5-deoxycytidylic acid, 5-deoxythymidylic acid, 5-deoxyguanylic acid, 5'-deoxyadenylic acid and other 5-deoxyribonucleotides. Such 5-nucle0tides as 5'-cytidylic acid or 5'- uridylic acid, which are produced by the hydrolysis of nucleic acid and are not employed as flavoring agents, are useful donors of the ribose-S'-phosphate radical for the synthesis of 5'-in0sini-c acid and 5'-guanylic acid which are useful seasoning agents.

The bacteria employed as an enzyme source can be prepared by submerged culture or stationary culture in synthetic or natural media at 20 to 40 C., for 15 to 50 hours. The enzyme source can be admixed to the reaction mixture in the form of a culture broth, as living cells, dried cells, or cell extracts.

The enzyme reaction of the present invention may be carried out in an aqueous medium. Insoluble organic bases dissolve in the solution as the reaction proceeds. The reaction proceeds in the pH range from to 10, and best between pH 7 and 9. The reaction may be carried out at temperatures from 30 to 60 C., and preferably from 40 to 50 C.

5-nucleotides synthesized according to the method of the invention may be isolated from the reaction mixture by such conventional methods as ion exchange chromatography or precipitation. After removal of bacterial cells, the reaction mixture may be passed over a column packed with an anion exchange resin in the (Cl) form (such as Dowex 1X4). The resin is then washed with water, the nucleotide adsorbed on the column is eluted by a suitable solvent, and the fractions containing the desired nucleotide are neutralized, concentrated, and the nucleotide is precipitated by alcohol. 5'-nucleotides are obtained as crude crystals of an alkali metal salt.

The nucleotides synthesized may be identified by conventional methods such as detection of the phosphate ester linkage, carbazole reaction, spectrophotometric metaperiodate oxidation, detection of inorganic phosphate liberated with snake venom 5-nucleotidase, or paper electrophoresis in acetic acid as a solvent. The nucleotide synthesized from organic bases by the method of this invention as described hereinafter were identified as 5'-isomers by the methods described above.

The following examples are further illustrative of the method of the invention, but it will be understood that the invention is not limited to the examples.

Example 1 20 ml. of a nutrient medium containing 1.0% meat extract, 1.0% peptone, 1.0% glucose, and 0.5% sodium chloride (pH 7.0) were placed in a 500 ml. shaking flask, and sterilized at 115 C. for 10 minutes. The medium was inoculated with Pseudomonas trifolii (ATCC-14537) and cultured aerobically at 30 C. for 20 hours. The bacterial cells were collected by centrifuging and suspended in distilled water.

800 ml. of an aqueous solution of 1.35 g. adenine and 14.3 g. disodium-5'-un'dylate, and having a ph of 9.0, were mixed with 200 ml. of the cell suspension which contained 3.5 g. dry cell material. The pH of the mixture was adjusted to 9.0, and it was incubated statically at 45 C. for 3 hours. The solution then contained 0.22 g./dl. 5'-adenylic acid (a yield of 66.1% based on the adenine originally present).

The bacterial cells were removed from the reaction solution by centrifuging, the supernatant liquid obtained was adjusted to pH 9.0, and the solution was passed over a column packed with 200 ml. of an anion exchange resin of the chloride type (Dowex 1X4). The column was washed with about 3 liters of water in which unreacted adenine was eluted, and then with 0.005 M acetate butler solution containing 0.146% soduim chloride (pH 5.5). The butler eluate was collected in 200 ml. fractions. Nucleosides were eluted first. Unreacted -uridylic acid was eluted in the fractions from 6 to 11.5 liters, and 5-adenylic acid was eluted in the fraction from to 40 liters. The combined 5-adenylic acid fractions were neutralized with sodium hydroxide solution, partly evaporated under reduced pressure, and precipitated sodium chloride was removed. An equal volume of ethanol was added to the concentrated solution, the mixture was cooled, and sodium 5'-adenyl ate was obtained as crude crystals weighing 2.16 g.

Example 2 A cell suspension of Pseudomonas trifolii ATCC.14537 was prepared as described in Example 1.

for 6 hours.

Nine 400 ml. batches of an aqueous solution, each batch containing 67.5 mg. adenine, 238 mg. disodium-5'- uridylate and 136 mg. potassium dihydrogen phosphate were respectively adjusted to pH values from 4 to 10 as listed below, and 10 ml. of the cell suspension 1 mg. dry cell substance) were added to each batch. The pH of each batch was again adjusted, and the reaction was permitted to proceed statically at 37 C. for 3 hours. The yield of the 5'-adenylic acid synthesized and the 5 '-uridylic acid decomposed are listed below.

5'-adenylic acid produced pH of the re- 5-uridylic acid action solution decomposed Concentration Yield (percent) (percent) (mg/ml.)

Note: 1/2 M-acetate bufier solution was used for adjusting the pH between 4 and 6. 1/2 M-Tris-hydrochloride bufier solution was used for a pH oi 7.0 to 10.0.

Example 3 40 ml. batches of an aqueous solution, each batch containing 67.5 mg. adenine, 715 mg. disodium-5-uridylate and 136.1 mg. potassium dihydrogen phosphate were mixed with 10 ml. of a cell suspension prepared as in Example 1 and containing 150 ml. all substance on a dry basis. The pH of the mixtures was adjusted to 9.0,

and they were incubated for 3 hours at the temperature listed below. 68.1 mg. of hypoxanthine and 75.6 mg. of

guanine were respectively substituted for the adenine in otherwise identical series of reactions. The yields of 5'-adenylic acid, 5'-inosinic acid, and 5'-guanylic acid are tabulated below.

A cell suspension of Alcaligenes metalcaligenes (AN-3) was prepared in the manner described in Example 1 and employed in converting 1.35 g. adenine as described in Example 1.

nally present adenine). In the reaction, the amino group in the 6-position of the adenine ring was deaminated by the action of the Alcaligenes metalcaligenes (AN-3). The reaction solution was worked up as described in Ex-.

ample 1, and 2.04 g. of crude crystalline sodium-5'.- inosinate were obtained.

Example 5 A cell suspension of Pseudomonas trifolii (ATCC- 12287) was prepared as described in Example 1.

An aqueous solution containing 1.36 g. hypoxanthine, and 14.3 g. disodium-5'-uridylate was treated with the cell suspension as described hereinabove at 4045 C. The reaction solution then contained 0.22 g./dl. 5-inosinic acid (63.5% based on the hypoxanthine).

A concentration of 0.18 g./dl. .5'-inosinic acid was obtained (51.7% based on the origi- The solution was worked up, and 2.58 g. sodium-5- inosinate were obtained.

Example 6 A cell suspension of Pseudomonas trifolii ATCC12287 was prepared as described in Example 1.

1.51 grams guanine and 14.3 g. disodium-5-uridylate were reacted in the presence of the cell suspension of Ps. trifolii at 50 C. for '6 hours. The reaction mixture contained 0.09 g./dl. 5'-guanylic acid (24.7% based on original guanine). The reaction solution was worked up as described in Example 1, and 5-guanylic acid was eluted in the fractions from liters to 21 liters. From the combined fractions, 693 mg. s0dium-5-guanylate were obtained.

Example 7 Cell suspension of the bacteria listed in the following table was prepared as described in Example 1.

A cell suspension of Pseudomonas trifoliz (ATCC 14537) was prepared as described in Example 1.

Aqueous solutions were prepared to contain per ml. batch, 715 mg. disodium-5'-uridylate, 136.1 mg. potassium dihydrogen phosphate, and one of the following bases: 60.1 mg. purine, 99.1 mg. Z-amino purine (nitrate), 208.2 mg. 2,6-diaminopurine (sulfate), 64.5 mg. barbituric acid, 63.1 mg. thymine, 74.6 mg. 6-methylaminopurine, 81.6 mg. G-dimethylaminopurine, 113.2 mg. benzyladenine, 107.6 mg. 6-furfurylaminopurine. Each batch was mixed with 10 ml. of the cell suspension 150 mg. dry cell substance), and the mixture was incubated statically at pH 9.0 and at 4045 C. for 3-6 hours.

Inosinic acid Guanylic acid Xanthylic acid Bacteria Cone. Yield Cone. Yield Conc. Yield (mg/ml.) (percent) (mg./ml.) (percent) (mg/m1.) (percent) Ps. trifolii ATCC14537 2. 0 58. 4 0. 7 18. 2 0. 4 10. 8 Ps. perlurzda ATCC14536..- 1. 8 50. 8 0. 5 14. 1 0.2 5. 6 A10. metalcalz'ge'nes AN-3. 2. 3 65. 8 0. 1 2. 6 0. 1 2. 8 Alt. viscolactis ATCCS036 2. 0 58. 1 0. 1 2. 6 0. 1 2. 8 Ser. marcescens ATCC1422 0. 6 16. 3 0.2 5. 2 0.2 4. 8 Ser. marcescem ATCC1422 0.6 17. 5 O. 2 5. 4 0. 2 4. 9 Mic. caseolyticus ATCC8460 0. 2 5. 5 0.2 4. 8 0.2 4. 6 St. cilreus ATCC4012 0.2 5. 2 0.2 4. 7 0.2 4. 4 Flew. harriso'nii ATC C-l4589 0. 5 14. 9 0. 2 5. 0 0. l 2. 8 Flav. fuscum ATC C-14233. 0.1 2. 6 0.1 2. 3 0.1 2. 8 E. intermedia A-21 0. 2 5. l 0.1 2. 3 0. 1 2. 8 E. coli IAM-1239 ATCC- 0. 1 2. 5 Trace Trace Aerbact. uerogeues ATCC8329 0. 1 2. 5 Trace Trace Proteus mirabz'lis ATCC15290 0.3 8. 6 Trace Trace Proteus v'uZgaris YO-13 0. 1 2. 5 Ps. trijalii X-2-142 1. 7 47. 3 0. 4 0. 3 8. 1

Example 8 The concentrations and yields of the 5-nucleot1de Example 9 Cell suspensions of Pseudomonas trifolii (ATCC 12287) and Alcaligenes viscolactis (ATCC9036) were prepared as described in Example 1.

Aqueous solutions respectively containing 67.6 mg. adenine, 68.1 mg. hypoxanthine, 75.6 mg. guanine, and 76.1 mg. xanthine, and each containing 715 mg. disodium- 5-cytidylate were incubated with the cell suspensions as described in Example 7. 5'-nucelotides corresponding to synthesized are listed below.

Cone. Yield (mg/ml.) (percent) Purine-riboside-5-phosphate 0. 8 23. 8 2-aminopurine/rib0side-5-phosphate 1. 2 34. 8 2,6-diaminopurine'riboside-5'-phosphate. 1. 2 17. 1 Thymine-riboside-5-phosphate 1. 5 43. 4 Barbituric acid-riboside-5-ph0sphate 0.6 17.2 6-methylaminopurine-rib0side-5-phosphate. 2. 0 54. 5 6-dimethylaminopurineriboSide-5-phosphate 1. 5 39. 2 Benzyladenine-rib oside-5-phosphate 2. 1 47. 5 G-furfurylaminopurine-riboside-5-phosphate O. 9 20. 9

Example 11 Three reaction mixtures were prepared each from 40 ml. of a solution containing 588 mg. diammonium-5- thymidylate, 136.1 mg. of potassium dihydrogen phosphate and respectively containing 67.6 mg. adenine, 68.1 mg, hypoxanthine, and 75.6 mg. guanine, with 10 ml. of a cell suspension of Pseudomonas trifolii ATCC14537 mg. dry cell matter) prepared as described in Example 1, and the mixtures were incubated at pH 9.0 and at 40-45 C. for 3-6 hours. '-deoxynucleotides were obtained as follows:

Concentration Yield (mg./ml.) (percent) 5-deoxyadeny1ic acid 1. 9 58. 1 5-deoxyinosinic acid 1. 7 50. 7 Sf-deoxyguanylic acid 0. 7 19. 9

Example 12 Pseudomonas trifolii (ATCC-14537) was grown as described in Example 1, except that the culture medium contained 4% glucose, 0.1% KH PO 0.04% MgSO 7I-I O, 2 p.p.m Fe++, 2 ppm. Mn++, 0.5% amino acid mixture, 0.5 soybean meal hydrolysate, and 2% l-pyroglutamic acid (pH 7.7).

20 ml. of the culture medium obtained were added to 80 ml. of an aqueous solution which contained 252.2 mg. thymine and 1.96 g. disodium 5-inosinate. The pH of the mixture was adjusted to 9.0 and it was incubated at 45 to 50 C. for 20 hours. As the end of this period, 226 mg. thymine riboside-5-monophosphate (a yield of 33.5% based on the thymine initially present) had accumulated in the reaction mixture. It was evaporated to 30 ml., adjusted to pH 2.5, and 30 ml. ethanol were added.

Cells and other insoluble substances were removed by centrifuging. The pH value of the supernatant liquid was adjusted to 7.7,and it was stored at 5 C. overnight. Most of the 5'-inosinate still present (about 95%) was-crystallized thereby. The thymine riboside-5'-monophosphate was soluble in the mother liquor which was partly evaporated in vacuo. The concentrate was diluted fold with water, adjusted to pH 10.8, and passed through a column of an anion exchange resin, Duolite AlO2D (chloride ion type), which was then washed with four liters of water. The unreacted thymine was eluted first. The hypoxanthine formed was eluted with 18 liters of 0.01 M acetate bufifer (pH 5.5) containing 0.059% sodium chloride, and the thymine riboside-S'-monophosphate was obtained next by elution with 9 liters of a solution containing 0.29% sodium chloride in the same buffer. The residual 5'- inosinic acid was recovered last with 4 liters of 2-N hydrochloric acid. The eluate containing the thymine riboside-5-monophosphate was neutralized with sodium hydroxide, concentrated in vacuo, and filtered to remove inorganic salts. It was then diluted with alcohol.

193 mg. sodium 5-ribothymidylate were obtained as crude crystals.

While the invention has been described with particular reference to specific embodiments, it is to be understood that it is not limited thereto, but is to be construed broad- -ly and restricted solely by the scope of the appended claims.

What we claim is:

1. A process for preparing a 5'-nucleotide having a desired purine or pyrimidine base component, which com prises contacting an enzyme source with an aqueous medium containing the organic base corresponding to the desired base component and a 5-nucleotide other than that to be prepared and having a base component selected from the group consisting of a purine and pyrimidine which is different from said desired base component until the desired 5-nucleotide is formed; and recovering the formed 5'-nucleotide, said enzyme source being capable of producing a 5-nucleotide selected from the group consisting of adenylic acid and inosinic acid when in contact with an aqueous solution containing 0.135 g. adenine and 1.43 g. disodium-5'-uridylate at 40 C. fora period of 3 to 20 hours at pH 8 to 9, said enzyme source being prepared from a microorganism selected from the genera Alcaligenes, Escherichia, Aerobacter, Pseudomonas,

Staphylococcus, Serratia, Flavobacterium, Proteus, and.

Micrococcus.

2. A process as set forth in claim 1, wherein said. enzyme source is a cell material of a bacterium selected from the group consisting of Alcaligenes metacaligenes AN-3, Alcaligenes viscolactis ATCC-9036, Escherichia.

intermedia A-2l, Escherichia coli ATCC-15289, Aerobacter aerogencs ATCC-8329, Pseudomonas trifolii ATCC-l4537 and ATCC-12287, Pseudomonas perlurida ATCC-145 36, Staphylococcus citreus ATCC-40l2, Staphylococcus aureus IAM- 8, Serratia marcescens ATCC- and ATCC-14226, F lavobacterium harrisoni 1 14227 ATCC-l4589, F lavobacterium fuscum ATCC-14233, Flavobacterium odoratum ATCC-465l, Proteus mirabilis ATCC-15290, Proteus vulgaris YO-l3, Micrococcus caseolyticus ATCC-8460, Micrococcus varians ATCC- 399 and Pseudomonas trifolii mutant X-2-142.

3. A process as set forth in claim 2, wherein said organic base is a purine base.

4. A process as set forth in claim 3, wherein said purine base is a member of the group consisting of purine, adenine, hypoxanthine, xanthine, guanine, 2-aminopurine, 2, fi-diaminopurine, 6-methylaminopurine, 6-dimethylaminopurine, G-furfurylaminopurine, benzyladenine, and kinetin.

5. A process as set forth in claim 2, wherein said organic base is a pyrimidine base.

6. A process as set forth in claim 5, wherein said pyylic acid, 5'-cytidylic acid, and 5'-uridylic acid.

9. A process as set forth in claim 2, wherein said other 5'-nucleotide is a 5'-dcoxyribonucleotide.

10. A process as set forth in claim 9, wherein said 5'- deoxyribonucleotide is a member of the group consisting of 5-deoxycytidylic acid, 5'-deoxythymidylic acid, 5-deoxyguanylic acid, and 5-deoxyadenylic acid.

11. A process as set forth in claim 2, wherein said cell material includes living cells of said bacterium.

12. A process as set forth in claim 2, wherein the pH of said aqueous medium is between 5 and 10.

13. A process as set forth in claim 2, wherein the pH of said aqueous medium is between 7 and 9.

14. A process as set forth in claim 2, wherein said aqueous medium is at a temperature of 30 to 60 C.

15. A process as set forth in claim 2, wherein said aqueous medium is at a temperature of 40 to 50 C.

16. A process as set forth in claim 2, wherein said.

aqueous medium includes ions of phosphoric acid.

17. A process as set forth in claim 2, wherein said cell material essentially consists of living cells of said bacterium, said aqueous medium is at a pH between 5 and.

10 and at a temperature between substantially 30 and 60 C., said organic base is a member of the group consis-ting of purine, adenine hypoxanthine, xanthine, guanine, 2-aminopurine, 2,6-diaminopurine, 6-methylaminopurine, 6-dimethylaminopurine, 6-furfurylaminopurine, benzyladenine, and kinetin, uracil, S-hydroxymethyluracil, cytosine, thymine, and barbituric acid, and said other 5- nucleotide is a member of the group consisting of5' adenylic acid, 5'-inosinic acid, 5'-xanthylic acid, 5'-guanylic acid, 5'-cytidylic acid, and 5-uridylic acid, 5'-deoxycytidylic acid, 5'-deoxythymidylic acid, 5-deoxyguanylic acid, and 5'-deoxyadenylic acid.

No references cited.

A. LOUIS MONACELL, Primary Examiner.

ALVIN E. TANENHOLTZ, Examiner. 

1. A PROCESS FOR PREPARING A 5''-NUCLEOTIDE HAVING A DESIRED PURINE OR PYRIMIDINE BASE COMPONENT, WHICH COMPRISES CONTACTING AN ENZYME SORCE WITH AN AQUEOUS MEDIUM CONTAINING THE ORGANIC BASE CORRESPONDING TO THE DESIRED BASE COMPONENT AND A 5''-NUCLEOTIDE OTHER THAN THAT TO BE PREPARED AND HAVING A BASE COMPONENT SELECTED FROM THE GROUP CONSISTINGD OF A PURINE AND PYRIMIDINE WHICH IS DIFFERENT FROM SAID DESIRED BASE COMPONENT UNTIL THE DESIRED 5''-NUCLEOTIDE IS FORMED; AND RECOVERING THE FORMED 5''-NUCLEOTIDE, SAID ENZYME SOURCE BEING CAPABLE OF PRODUCING A 5''-NUCLEOTIDE SELECTED FROM THE GROUP CONSISTING OF ADENYLIC ACID AND INOSINIC ACID WHEN IN CONTACT WITH AN AQUEOUS SOLUTION CONTAINING 0.135 G. ADENINE AND 1.43 G. DISODIUM-5''- URIDYLATE AT 40*C. FOR A PERIOD OF 3 TO 20 HOURS AT PH 8 TO 9, SAID ENZYME SOURCE BEING PREPARED FROMS A MICROORGANISM SELECTED FROM THE GENERA ALCALIGENES, ESCHERICHIA, AEOBACTER, PSEUDOMONAS, STAPHYLOCOCCUS, SERRATIA, FLAVOBACTERIUM, PROTEUS, AND MICROCOCCUS. 